Abstract An alternative method devised to isolate functionally active antibody F(ab′) 2 fragments required fewer manipulations and used less serum than do methods generally used. The method involved pepsin digestion of the whole globulin fraction precipitated from as little as 3 ml of serum. Chromatographic separation of the digest on Sephadex G-150 yielded two distinct peaks: Peak I consisted of lipoprotein and showed no immunoglobulin determinants; Peak II, as established by immunodiffusion analysis, contained immunoglobulin F(ab′) 2 fragments. Radioimmunoassays performed on Peak II protein to determine the presence of allotypic markers revealed less than 10% contamination by non-immunoglobulin protein; coprecipitation tests, used to characterize Peak II further, also showed less than 10% contamination by non-immunoglobulin protein. Recovery of total serum IgG F(ab′) 2 was 90% or greater using this technique, compared with a potential 20–25% recovery using standard isolation procedures.